Methods for specifically amplifying target nucleic acids have become very important techniques in molecular biological research and in clinical applications including genetic testing.
The most widely used nucleic acid amplification method is the PCR (polymerase chain reaction) method, but this requires complex temperature control. Therefore, the LAMP (loop-mediated isothermal amplification) method, the ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids) method and other isothermal amplification methods have also been developed.
Nucleic acids amplified by the PCR, LAMP and ICAN methods and the like are double strands, and methods using fluorescent dyes are being actively studied as detection methods for them.
The most common method is a method of performing agarose electrophoresis on a solution after the amplification reaction, followed by binding to a fluorescent intercalator such as ethidium bromide or SYBR Green and then observing the specific fluorescence (Non Patent Literature 1). However, the method of detection using a fluorescent intercalator after electrophoresis requires an electrophoresis running time of about 30 minutes to an hour, as well as expensive equipment such as a UV irradiation device or fluorescence detection device for detecting the fluorescence.
If there is no possibility of other DNAs being present and only the presence or absence of the amplified product needs to be determined, the electrophoresis step can be omitted by adding beforehand the fluorescent intercalator to the reaction liquid before the PCR reaction, and then detecting the fluorescence after the amplification reaction (Patent Literature 1). However, the problem is that since fluorescent intercalators bind to primers and other single-stranded nucleic acids, the background signals are then amplified, thereby leading to reduced detection sensitivity. In this context, a method has been developed for reducing background signals by treating with a compound that reacts preferentially with a fluorescent intercalator bound to a single-stranded nucleic acid (Patent Literature 2), and fluorescent dyes have been developed having improved reaction specificity to double-stranded nucleic acids compared to single-stranded nucleic acids (Patent Literature 3), but in both cases fluorescent detection is still used.
Alternatively, a method has been developed for detection by fluorescence polarization by performing a nucleic acid amplification reaction using a fluorescent labeled primer (Patent Literature 4), but in general fluorescent primers are expensive, and the method is also disadvantageous, for example, in that it is complex because it requires an operation to separate out the fluorescent labeled primer not incorporated into the amplified product.